Neutrophils (PMN) destroy bacteria and fungi by several mechanisms, one of which depends on the presence of antibacterial peptides and proteins in the cell's azurophil granules and their delivery to phagolysosomes. Prominent among these antibacterial components are several small (Mr=3500) peptides, termed """"""""defensins,"""""""" which constitute 30-50% of the total protein in human azurophil granules. Although much information about defensin structure now exists, neither their antimicrobial mechanisms nor their overall roles in PMN function are known with certainty. The five specific aims of this proposal are 1: to characterize the effects of defensins on microorganisms and model membranes; 2. to examine the interactions of defensins with other components of PMN granules and with products of the respiratory burst; 3. to seek evidence that defensins operate within intact human leukocytes; 4. to compare human defensins with other antimicrobial or cytotoxic peptides and proteins, including two peptides, major basic protein (MBP) and eosinophil cationic protein (ECP), that are prominent in eosinophils; and 5. to screen for PMN defensin-deficiencies and to seek the presence of defensins and defensin-synthesis in cells other than PMN. More specifically, we will purify HNP-1, HNP-2 and HNP-3 from normal human PMN and test their in vitro activity against staphylococci, enterobacteria and yeast-phase Candida albicans. Defensins will also be combined with other proteins or peptides from human PMN granules to test for synergistic combinations. In addition, the interactions of defensins with OC1- and a myeloperoxidase, hydrogen peroxide, halide system will be examined to determine if defensins form reactive N-chloro groups that increase their antimicrobial efficacy. We will also characterize the uptake of defensins by susceptible and resistant microbial targets. Human defensins exert optimal microbicidal activity against bacteria and fungi that are metabolically active. They are inactive against respiratory-deficient C. albicans or nongrowing bacteria. Defensins cause sequential permeabilization of the outer membrane (OM) and inner membrane (IM) of a susceptible target, E. coli ML-35(pBR322). Such observations are consistent with and suggestive of the possibility that defensins form voltage-dependent membrane pores. We will use intact microorganisms and model membrane systems to test this hypothesis. The long term objective of this work is to discover and characterize the effector substances and antimicrobial mechanisms of phagocytic cells. Successful completion of this proposal will lead to a better understanding of host resistance mechanisms, and may facilitate the design of novel antibiotics patterned after defensins.
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